1344

A macrolactonization approach to the total synthesisof the antimicrobial cyclic depsipeptide LI-F04a

and diastereoisomeric analoguesJames R. Cochrane, Dong Hee Yoon, Christopher S. P. McErlean

and Katrina A. Jolliffe*

Full Research Paper Open Access

Address:School of Chemistry, The University of Sydney, 2006, NSW, Australia;Tel: +61-2-93512297; Fax: +61-2-93513329

Email:Katrina A. Jolliffe* - kate.jolliffe@sydney.edu.au

* Corresponding author

Keywords:antifungal; cyclic depsipeptide; epimerization; lipopeptide;macrolactonization; peptides

Beilstein J. Org. Chem. 2012, 8, 1344–1351.doi:10.3762/bjoc.8.154

Received: 02 May 2012Accepted: 16 July 2012Published: 21 August 2012

This article is part of the Thematic Series "Antibiotic and cytotoxicpeptides".

Guest Editor: N. Sewald

© 2012 Cochrane et al; licensee Beilstein-Institut.License and terms: see end of document.

AbstractThe cyclic peptide core of the antifungal and antibiotic cyclic depsipeptide LI-F04a was synthesised by using a modified

Yamaguchi macrolactonization approach. Alternative methods of macrolactonization (e.g., Corey–Nicolaou) resulted in significant

epimerization of the C-terminal amino acid during the cyclization reaction. The D-stereochemistry of the alanine residue in the

naturally occurring cyclic peptide may be required for the antifungal activity of this natural product.

1344

IntroductionThe LI-F or fusaricidin class of cyclic depsipeptides are

produced by a number of strains of Bacillus (Paenebacillus)

and exhibit antifungal and antibacterial activity against a range

of clinically relevant species, including Candida albicans,

Cryptococcus neoformans, Staphylococcus aureus and Micro-

coccus luteus [1-7]. These compounds have a cyclic hexadep-

sipeptide core, in which three amino acids, L-Thr, D-allo-Thr

and D-Ala are conserved throughout the series, while there are

slight variations in the other three amino acids. In LI-F04a these

are D-Asn, L-Val and D-Val. A unique 15-guanidino-3-

hydroxypentadecanoyl (GHPD) side chain is appended to the

cyclic peptide core through the nitrogen atom of the L-Thr

residue. There has been recent interest in the synthesis of the

LI-F family of cyclic depsipeptides due to their antifungal

activity. Biosynthetic processes have been employed to this end,

although these provide mixtures of depsipeptides, which makes

it difficult to determine structure–activity relationships [7,8].

More recently, the solid-phase synthesis of a number of

Beilstein J. Org. Chem. 2012, 8, 1344–1351.

1345

analogues of the fusaricidins has been reported. However, in all

cases, the side chain 3-hydroxy group was not incorporated into

the structure [9]. By total synthesis of both side-chain epimers

of this structure we have recently established that the absolute

configuration of this side-chain hydroxy group is (R) in the

naturally occurring LI-F04a 1 [10]. We employed a late-stage

coupling of the cyclic peptide core 2 with the GHDP side chain

3 to enable ready access to both side-chain epimers (Scheme 1).

While macrocyclization to give the core 2 could be performed

at any of the amide or ester bonds [10], we chose to use a

macrolactonization approach to enable ready access to

analogues of the LI-F04a core through straightforward Fmoc

solid-phase peptide synthesis of the linear precursors. We report

here our optimization of these macrolactonization conditions,

together with the synthesis of several analogues of LI-F04a

using this approach, and an investigation of the antifungal

activity of these synthetic lipodepsipeptides.

Results and DiscussionThe required linear precursor for the synthesis of 2 by a macro-

lactonization approach is 4 (Scheme 1), in which the N-terminal

amino group of the L-Thr residue is protected while the side-

chain hydroxy group is free. The Cbz group was chosen as a

suitable protecting group for the N-terminus. The D-Asn and

D-allo-Thr residues were the only amino acids requiring side-

chain protection. Given previous reports that 2,2-dimethylated

pseudoprolines (ΨMe,Me'Pros) [11-13] are useful turn-inducers

for improving yields of macrolactamization reactions [14-17],

we chose to prepare two linear precursors, 5 and 6 (Scheme 2),

in which the D-allo-Thr protecting group was either tert-butyl

or ΨMe,Me'Pro, respectively, to investigate whether a turn-

inducer might assist the macrolactonization reaction. Linear

peptides 5 and 6 were prepared by standard Fmoc solid-phase

peptide synthesis protocols using PyBOP/Hünigs base as the ac-

tivation reagent and 2-chlorotritylchloride resin to allow

cleavage of the peptide from the solid support with side-chain

protecting groups intact. In the case of 6, the ΨMe,Me'Pro was

introduced by coupling the known dipeptide Fmoc-Val-D-a-

Thr(ΨMe,Me'Pro)-OH [18] into the growing peptide chain.

While there are a large number of methods available for macro-

lactonization reactions, those most commonly employed include

the Corey–Nicolaou [19], Boden–Keck [20] and Yamaguchi

[21] lactonization procedures. We initially chose to screen these

three procedures for the macrolactonization of 5. In all three

cases (Table 1, entries 1–3), analysis of the crude product

mixtures showed that mixtures of cyclic diastereoisomers were

obtained, indicating that the C-terminal amino acid underwent

epimerization during the macrolactonization reactions (see

Supporting Information File 1 for full experimental details)

[22]. However, the ratio of the two diastereoisomers differed

Scheme 1: Retrosynthetic strategy.

significantly under the three sets of conditions, with the major

diastereomer formed under the Yamaguchi conditions differing

from the major product obtained in the other reactions.

In order to establish which set of conditions gave the highest

ratio of the desired product 7, the mixture of cyclic depsipep-

tides 7 and 8 obtained from macrolactonization under the

highest yielding conditions (Corey–Nicolaou) was separated,

and the major diastereomer was subjected to hydrolysis upon

Beilstein J. Org. Chem. 2012, 8, 1344–1351.

1346

Scheme 2: Macrolactonization reactions of seco acids 5 and 6 (for reagents and yields see Table 1 and Table 2).

Table 1: Reaction conditions for macrocyclization of 5.

entry reaction conditions yield of major isomera ratio of 7:8b

1 dithiopyridine, triphenylphosphine, MeCN, 80 °C 56% 13:872 DCC, DMAP, camphorsulfonic acid, CH2Cl2 14% 45:553 2,4,6-trichlorobenzoyl chloride, DMAP, NEt3, toluene, 110 °C 20% 61:394 2,4,6-trichlorobenzoyl chloride, DMAP, iPr2NEt, toluene, 80 °C 33% 69:315 2,4,6-trichlorobenzoyl chloride, DMAP, NEt3, toluene, 25 °C 33% 89:116 2,4,6-trichlorobenzoyl chloride, DMAP, NEt3, THF, 25 °C 16% 82:187 2,4,6-trichlorobenzoyl chloride, DMAP, NEt3, toluene, 25 °C, slow addition of 5 58% 92:8c

8 2-methyl-6-nitrobenzoic anhydride, DMAP, NEt3, toluene, 25 °C, slow addition of 5 52% 94:69 cyanuric chloride, MeCN, 25 °C 30% 80:20

aIsolated yield; bas determined by integration of analytical HPLC traces of the crude reaction mixtures; caverage value (5–12% epimerization wasobserved upon repeat reactions).

treatment with MeOH/H2O/aq NH4OH (4:1:1 v/v/v). Compari-

son of the crude peptide, so obtained, to authentic samples of

both 5 and the C-terminal epimer 9 (which was independently

prepared by solid-phase peptide synthesis), indicated that the

major product obtained upon macrolactonization of 5 under the

Corey–Nicolaou conditions was the undesired cyclic

depsipeptide 8, in which the C-terminal Ala residue had epimer-

ized (Figure 1). Since the Yamaguchi conditions gave impro-

ved ratios of the desired/epimerized cyclic depsipeptides,

subsequent optimization of the macrolactonization conditions

focussed on this and related procedures (Table 1). The best

yields of the desired cyclic depsipeptide 7 (58% isolated yield,

with 5–12% of epimer 8 also observed) were obtained using a

modification of Yonemitsu’s conditions [23] in which linear

peptide 5 was added slowly to a solution of DMAP, 2,4,6-

trichlorobenzoyl chloride and triethylamine in toluene at room

temperature. Similar yields and low epimerization (52% isol-

ated yield, 6% epimer observed) were obtained using 2-methyl-

6-nitrobenzoic anhydride (MNBA) [24] as the activating agent

in place of 2,4,6-trichlorobenzoyl chloride.

Beilstein J. Org. Chem. 2012, 8, 1344–1351.

1347

Figure 1: Analytical HPLC traces of linear peptides. (a) Compound 9 (retention time = 31.5 min); (b) Compound 5 (retention time = 30.7 min); (c)co-injection of mixture of 9 and 5 (2:1); (d) Crude reaction mixture after hydrolysis of major cyclic peptide from Corey–Nicolaou cyclization, indicatingthat the major product of hydrolysis is 9; ▲ indicates the unreacted cyclic peptide (retention time = 34.0 min) and ● is a peak attributed to hydrolysis ofboth the cyclic peptide ester bond and Cbz protecting group (LCMS m/z = 902 [M + H]+).

To investigate whether the ΨMe,Me'Pro would assist macrolac-

tonization, linear precursor 6 was subject to cyclization under a

range of conditions (Table 2). However, in all cases, the cycliz-

ation yields were found to be lower for the ΨMe,Me'Pro-

containing peptide than for the tert-butyl protected precursor 5,

with higher amounts of C-terminal epimerization also observed,

except in the case of cyclization using cyanuric chloride [25].

Unfortunately, the yield could not be improved above 17% in

this case, so all further reactions were performed using the

Thr(O-t-Bu) protected cyclic peptides 7 and 8. With cyclic

peptides 7 and 8 in hand, we chose to attach the GHPD side

chain to both compounds to enable the effect of peptide stereo-

chemistry on the biological activity of the LI-F cyclic peptides

to be assessed. Additionally, to investigate the effect of the

3-hydroxy group of the GHPD side chain on the biological

activity of this class of cyclic peptide, we prepared the dehy-

droxy side-chain analogue 12 for attachment to the cyclic

peptide core (Scheme 3). Notably, a previously synthesised

Beilstein J. Org. Chem. 2012, 8, 1344–1351.

1348

Table 2: Reaction conditions for macrocyclization of 6.

entry reaction conditions yield of major isomera ratio of 10:11b

1 dithiopyridine, triphenylphosphine, MeCN, 80 °C 24% <5 to >952 2,4,6-trichlorobenzoyl chloride, DMAP, NEt3, toluene, 110 °C 19% 50:503 2,4,6-trichlorobenzoyl chloride, DMAP, NEt3, toluene, 25 °C 7% 80:204 2,4,6-trichlorobenzoyl chloride, DMAP, NEt3, toluene, 25 °C, slow addition of 5 24% 79:215 cyanuric chloride, MeCN, 25 °C 17% >95 to <5

aIsolated yield; bas determined by integration of analytical HPLC traces of the crude reaction mixtures.

LI-F04a analogue with a twelve-carbon side chain lacking the

hydroxy group has been observed to have antimicrobial activity

[9]. Thus, hydrolysis of pentadecanolide 13 [26] was followed

by esterification to give the methyl ester 14 in excellent yield.

Reaction of 14 with di(tert-butoxycarbonyl)guanidine under

Mitsunobu conditions [27] proceeded smoothly to give 15 in

86% yield. Hydrolysis of the methyl ester followed by acidic

work up to enable extraction of the resulting carboxylic acid

gave 12, in which one of the guanidino Boc protecting groups

was also removed.

Scheme 3: Synthesis of the dehydroxy side chain 12.

Hydrogenolysis of the Cbz protecting groups of 7 and 8 gave

the corresponding amines 16 and 17, respectively (Scheme 4).

These were coupled with the previously synthesised side chains

18, 19 [10] and 12, by using HATU as the coupling agent. The

resulting compounds were not isolated but immediately

subjected to global deprotection upon treatment with trifluoro-

acetic acid/CH2Cl2/H2O (90:5:5 v/v/v) to give LI-F04a (1),

side-chain epimer 20, dehydroxy analogue 21 and the two side-

chain epimers of the L-Ala derivative, 22 and 23.

The antifungal activity of 1 and 20–23 was evaluated by using a

standardised serial dilution sensitivity assay [28] against refer-

ence strains of Candida albicans, Cryptococcus neoformans and

Aspergillus fumigatus (Table 3). Synthetic LI-F04a was found

to exhibit good activity against both C. albicans and C.

neoformans, but only modest activity against A. fumigatus,

consistent with the previously reported activity of the natural

product [1,3,5]. The side-chain epimer 20 exhibited signifi-

cantly lower activity than 1 against C. albicans and C.

neoformans, indicating that the stereochemistry of the side-

chain hydroxy group is important for the antifungal activity of

the compounds. Removal of the hydroxy group, as in 21,

resulted in a further small decrease in activity against these

species. Notably, compounds 22 and 23, prepared from the

C-terminal-epimerised cyclic peptide did not exhibit antifungal

activity against any of the species tested. This suggests that the

conformation of the cyclic peptide core is important in deter-

mining the antifungal activity of these compounds, since inver-

sion of the stereocentre of one of the amino acids in the macro-

cycle is expected to result in a significantly different peptide

conformation [29]. Modelled structures of the side-chain-acyl-

ated cyclic peptides 24 and 25 obtained by using Monte Carlo

conformational searches in Macromodel [30] suggest that these

cyclic peptides adopt significantly different conformations with

different arrangements of hydrogen bonds (Figure 2). The

temperature dependence of the chemical shifts of the signals

attributable to the amide NHs of 1 in d6-DMSO was deter-

mined experimentally and confirmed the involvement of the

D-Ala and D-Asn amide protons in hydrogen bonds [31] as

suggested by the modelling studies of the acetamide analogue

(see Supporting Information File 1 for details). This indicates

that these hydrogen bonds may be important in locking the

cyclic depsipeptide into a biologically active conformation.

Beilstein J. Org. Chem. 2012, 8, 1344–1351.

1349

Scheme 4: Synthesis of LI-F04a (1) and analogues 20–23.

Table 3: Antifungal activity.

compoundC. albicansATCC 10231(MIC µM)a

C. neoformansATCC 90112(MIC µM)a

A. fumigatusATCC 204305(MIC µM)a

1 5.5 2.8 4420 22 11 2221 44 22 2222 >88 >88 >8823 >88 >88 >88

aMICs for amphotericin B: C. albicans 0.35 µM; C. neoformans 0.35 µM; A. fumigatus 0.70 µM.

Beilstein J. Org. Chem. 2012, 8, 1344–1351.

1350

Figure 2: Structures and lowest-energy conformers of 24 (left) and 25 (right) obtained using Macromodel. Hydrogen bonding is highlighted in yellow.

ConclusionIn summary, macrolactonization to form the cyclic depsipeptide

core of LI-F04a was achieved in good yields by using either

modified Yonemitsu conditions or similar conditions, in which

the 2,4,6-trichlorobenzoyl chloride activating agent was

replaced with MNBA. Slow addition of the linear seco-acid to

the activating agents was found to be the key factor to minim-

izing epimerization of the C-terminal amino acid during the

macrolactonization reaction. Synthetic LI-F04a was found to

exhibit similar antifungal activity to that reported for the natur-

ally occurring material. The antifungal activity of 1 was reduced

upon either inversion of the stereochemistry, or deletion of the

side-chain hydroxy group. Inversion of the D-Ala residue in the

cyclic depsipeptide core resulted in complete loss of antifungal

activity, indicating that the cyclic peptide conformation may be

important in the biological activity of this class of cyclic

lipodepsipeptide.

Supporting InformationSupporting Information File 1Experimental details for all new compounds.

[http://www.beilstein-journals.org/bjoc/content/

supplementary/1860-5397-8-154-S1.pdf]

Supporting Information File 21H, 13C and 2D NMR data for all new compounds.

[http://www.beilstein-journals.org/bjoc/content/

supplementary/1860-5397-8-154-S2.pdf]

AcknowledgementsWe thank the University of Sydney for financial support and the

award of a postgraduate scholarship to JRC; and Prof. Tania

Sorrell and Dr. Julianne Djordjevic (Centre for Infectious

Beilstein J. Org. Chem. 2012, 8, 1344–1351.

1351

Diseases and Microbiology, Westmead Hospital, University of

Sydney) for assistance with the antifungal assays.

References1. Kurusu, K.; Ohba, K.; Arai, T.; Fukushima, K. J. Antibiot. 1987, 40,

1506–1514. doi:10.7164/antibiotics.40.15062. Kajimura, Y.; Sugiyama, M.; Kaneda, M. J. Antibiot. 1995, 48,

1095–1103. doi:10.7164/antibiotics.48.10953. Kajimura, Y.; Kaneda, M. J. Antibiot. 1996, 49, 129–135.

doi:10.7164/antibiotics.49.1294. Kajimura, Y.; Kaneda, M. J. Antibiot. 1997, 50, 220–228.

doi:10.7164/antibiotics.50.2205. Kuroda, J.; Fukai, T.; Konishi, M.; Uno, J.; Kurusu, K.; Nomura, T.

Heterocycles 2000, 53, 1533–1549. doi:10.3987/COM-00-89226. Kaneda, M.; Kajimura, Y. Yakugaku Zasshi 2002, 122, 651–671.7. Choi, S.-K.; Park, S.-Y.; Kim, R.; Lee, C.-H.; Kim, J. F.; Park, S.-H.

Biochem. Biophys. Res. Commun. 2008, 365, 89–95.doi:10.1016/j.bbrc.2007.10.147

8. Li, J.; Beatty, P. K.; Shah, S.; Jensen, S. E. Appl. Environ. Microbiol.2007, 73, 3480–3489. doi:10.1128/AEM.02662-06

9. Bionda, N.; Stawikowski, M.; Stawikowska, R.; Cudic, M.;López-Vallejo, F.; Treitl, D.; Medina-Franco, J.; Cudic, P.ChemMedChem 2012, 7, 871–882. doi:10.1002/cmdc.201200016

10. Cochrane, J. R.; McErlean, C. S. P.; Jolliffe, K. A. Org. Lett. 2010, 12,3394–3397. doi:10.1021/ol101254m

11. Wöhr, T.; Wahl, F.; Nefzi, A.; Rohwedder, B.; Sato, T.; Sun, X.;Mutter, M. J. Am. Chem. Soc. 1996, 118, 9218–9227.doi:10.1021/ja961509q

12. Dumy, P.; Keller, M.; Ryan, D. E.; Rohwedder, B.; Wöhr, T.; Mutter, M.J. Am. Chem. Soc. 1997, 119, 918–925. doi:10.1021/ja962780a

13. Keller, M.; Sager, C.; Dumy, P.; Schutkowski, M.; Fischer, G. S.;Mutter, M. J. Am. Chem. Soc. 1998, 120, 2714–2720.doi:10.1021/ja973966s

14. Skropeta, D. S.; Jolliffe, K. A.; Turner, P. J. Org. Chem. 2004, 69,8804–8809. doi:10.1021/jo0484732

15. Sayyadi, N.; Skropeta, D.; Jolliffe, K. A. Org. Lett. 2005, 7, 5497–5499.doi:10.1021/ol0522891

16. Fairweather, K. A.; Sayyadi, N.; Luck, I. J.; Clegg, J. K.; Jolliffe, K. A.Org. Lett. 2010, 12, 3136–3139. doi:10.1021/ol101018w

17. Wong, M. S. Y.; Jolliffe, K. A. Aust. J. Chem. 2010, 63, 797–801.doi:10.1071/CH09643

18. Clegg, J. K.; Cochrane, J. R.; Sayyadi, N.; Skropeta, D.; Turner, P.;Jolliffe, K. A. Aust. J. Chem. 2009, 62, 711–719. doi:10.1071/CH09151

19. Corey, E. J.; Nicolaou, K. C. J. Am. Chem. Soc. 1974, 96, 5614–5616.doi:10.1021/ja00824a073

20. Boden, E. P.; Keck, G. E. J. Org. Chem. 1985, 50, 2394–2395.doi:10.1021/jo00213a044

21. Inanaga, J.; Hirata, K.; Saeki, H.; Katsuki, T.; Yamaguchi, M.Bull. Chem. Soc. Jpn. 1979, 52, 1989–1993. doi:10.1246/bcsj.52.1989

22. Atherton, E.; Sheppard, R. C. Solid Phase Peptide Synthesis: APractical Approach; IRL Press: Oxford, New York, 1989.

23. Hikota, M.; Sakurai, Y.; Horita, K.; Yonemitsu, O. Tetrahedron Lett.1990, 31, 6367–6370. doi:10.1016/S0040-4039(00)97066-7

24. Shiina, I.; Kubota, M.; Ibuka, R. Tetrahedron Lett. 2002, 43,7535–7539. doi:10.1016/S0040-4039(02)01819-1

25. Venkataraman, K.; Wagle, D. R. Tetrahedron Lett. 1980, 21,1893–1896. doi:10.1016/S0040-4039(00)92809-0

26. Nesmeyanov, A. N.; Zakharkin, L. I.; Kost, T. A.; Friedlina, R. K.Russ. Chem. Bull. 1960, 9, 195–199. doi:10.1007/BF00942889

27. Dodd, D. S.; Kozikowski, A. P. Tetrahedron Lett. 1994, 35, 977–980.doi:10.1016/S0040-4039(00)79943-6

28. Reference Method for Broth Dilution Susceptibility Testing of Yeasts:Approved Standard. NCCLS document M27-A (ISBN 1-56238-186-5).National Committee for Clinical Laboratory Standards, Pennsylvania,USA, 1997; Reference Method for Broth Dilution Susceptibility Testingof Filamentous Fungi: Approved Standard. NCCLS document M38-A(ISBN 1-56238-186-5). National Committee for Clinical LaboratoryStandards, Pennsylvania, USA, 2002.

29. Haubner, R.; Finsinger, D.; Kessler, H. Angew. Chem., Int. Ed. Engl.1997, 36, 1374–1389. doi:10.1002/anie.199713741

30. Mohamadi, F.; Richards, N. G. J.; Guida, W. C.; Liskamp, R.;Lipton, M.; Caufield, C.; Chang, G.; Hendrickson, T.; Still, W. C.J. Comput. Chem. 1990, 11, 440–467. doi:10.1002/jcc.540110405

31. Kessler, H. Angew. Chem., Int. Ed. Engl. 1982, 21, 512–523.doi:10.1002/anie.198205121

License and TermsThis is an Open Access article under the terms of the

Creative Commons Attribution License

(http://creativecommons.org/licenses/by/2.0), which

permits unrestricted use, distribution, and reproduction in

any medium, provided the original work is properly cited.

The license is subject to the Beilstein Journal of Organic

Chemistry terms and conditions:

(http://www.beilstein-journals.org/bjoc)

The definitive version of this article is the electronic one

which can be found at:

doi:10.3762/bjoc.8.154